Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

Background

Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14^th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.

Results

Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion

The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0086-y) contains supplementary material, which is available to authorized users.     

G Protein-regulated inducer of neurite outgrowth (GRIN) modulates Sprouty protein repression of mitogen-activated protein kinase (MAPK) activation by growth factor stimulation

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.     

Retention of plastic-tipped dart tags in African tigerfish Hydrocynus vittatus

Estimates of tag retention and tagging-related mortality are essential for mark-recapture experiments. Mortality and tag loss were estimated from 15 tigerfish Hydrocynus vittatus marked using Hallmark model PDL plastic-tipped dart tags released into a 1 730 m² pond at Kamutjonga Inland Fisheries Institute, Namibia, and inspected bi-monthly for the presence or absence of tags. No mortality was observed during the experiment. All marked fish had lost their tags after 10 months and 50% tag loss was estimated at 3.9 months. The high tag loss rate indicates that PDL plastic-tipped dart tags are not suitable for long-term studies on this species.     

High Rates of Ofloxacin Resistance in Mycobacterium tuberculosis among Both New and Previously Treated Patients in Tamil Nadu,South India

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.     

Cytotoxic and membrane perturbation effects of a novel amyloid forming model peptide poly(leucine-glutamic acid)

In the present study we have elucidated the toxicity of a novel amyloid forming model peptide, Poly (leucine-glutamic acid). The toxicity of the fibrils prepared from this peptide was analyzed in peripheral blood lymphocytes (PBL). The MTT reduction assay revealed that the viability of PBL decreases significantly upon treatment with Poly(leucine-glutamic acid) (Poly [LE]). Enhanced DCFH-DA fluorescence in treated cells suggests that peptide toxicity is probably mediated by the formation of free radicals. In vivo and in vitro biochemical studies indicated that Poly [LE] inactivates the antioxidant system of cells. Perturbation of Poly [LE] in a membrane lipid environment was assessed by circular dichroism (CD) using phosphotidyl choline-cholesterol bilayers. The CD results revealed that LE enhances its beta sheet content in a bilayer environment. Sequestration of Poly [LE] in lipid rafts demonstrates that it has a binding cleft similar to Abeta in lymphocyte raft domains. Nuclear membrane binding studies showed that Poly [LE] binds to nuclear membranes and may cause genotoxicity.     

Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism

Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells.     

Role of the chemical interactions of the agonist in controlling alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Structures of the isolated ligand binding domain of this receptor have provided significant insight into the large-scale conformational changes, which when propagated to the channel segments leads to receptor activation. However, to establish the role of specific molecular interactions in controlling fine details such as the magnitude of the functional response, we have used a multiscale approach, where changes at specific moieties of the agonists have been studied by vibrational spectroscopy, while large-scale conformational changes have been studied using fluorescence resonance energy transfer (FRET) investigations. By exploiting the wide range of activations by the agonists, glutamate, kainate, and AMPA, for the wild type and Y450F and L650T mutants of the GluR2 subtype, and by using the multiscale investigation, we show that the strength of the interactions at the alpha-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the alpha-amine group forms bridging interactions at the cusp of the ligand binding cleft, this appears to be a critical interaction through which the agonist controls the extent of activation of the receptor.